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The revolutionary insensitive, green, and healthier training technology with reduced adverse contamination (RIGHTTRAC) program is a 5-year technology demonstration program (TDP) aimed at showing that green and insensitive munitions have better properties than current munitions, and that it is feasible to implement safer weapon solutions that would ease the environmental pressure on ranges and training areas and decrease the health hazards for the users. The goals of this TDP are to reach a near-zero dud rate and to eliminate the potential for research development explosive (RDX) contamination as well as the use of toxic and carcinogenic compounds. This will be done by performing significant improvements to the fuzing system, the main explosive charge and the gun propellant. The vehicle used for this demonstration is a 105-mm army artillery munition (high-explosive M1), currently filled with Composition B (a 60:40 RDX:TNT mix) and using a single-base gun propellant (M1 formulation). The aim of this communication is to provide an overview of the project and to describe the steps taken to evaluate the environmental properties of the chosen propellant and explosive formulations. At its completion, it is believed that this project will have provided the required data to assess the long-term effects of the munitions on the environment right at an early stage of the weapon development cycle, and that this can be cost effective in the long run. This will help sustain military training while preserving our resources, as well as shaping the future of weapon system development.

The high explosive nitroaromatic 2,4-dinitroanisole (DNAN) is less shock sensitive than 2,4,6-trinitrotoluene (TNT), and is proposed as a TNT replacement for melt-cast formulations. Before using DNAN in munitions and potentially leading to environmental impact, the present study examines the ecotoxicity of DNAN using selected organisms. In water, DNAN decreased green algae Pseudokirchneriella subcapitata growth (EC50 = 4.0mg/L), and bacteria Vibrio fischeri bioluminescence (Microtox, EC50 = 60.3mg/L). In soil, DNAN decreased perennial ryegrass Lolium perenne growth (EC50 =7 mg/kg), and is lethal to earthworms Eisenia andrei (LC50 = 47 mg/kg). At sub-lethal concentrations, DNAN caused an avoidance response (EC50 = 31 mg/kg) by earthworms. The presence of DNAN and 2-amino-4-nitroanisole in earthworms and plants suggested a role of these compounds in DNAN toxicity. Toxicity of DNAN was compared to TNT, tested under the same experimental conditions. These analyses showed that DNAN was equally, or even less deleterious to organism health than TNT, depending on the species and toxicity test. The present studies provide baseline toxicity data to increase the understanding of the environmental impact of DNAN, and assist science-based decision makers for improved management of potential DNAN contaminated sites.

Nitroguanidine (NQ) is an energetic material that is used as a key ingredient of triple-base propellants and is currently being considered as a TNT replacement in explosive formulations. NQ was efficiently degraded in aerobic microcosms when a carbon source was added. NQ persisted in unamended microcosms or under anaerobic conditions. An aerobic NQ-degrading bacterium, Variovorax strain VC1, was isolated from soil microcosms containing NQ as the sole nitrogen source. NQ degradation was inhibited in the presence of a more favorable source of nitrogen. Resting cells of VC1 degraded NQ effectively (54 μmol h(-1) g(-1) protein) giving NH(3) (50.0%), nitrous oxide (N(2)O) (48.5%) and CO(2) (100%). Disappearance of NQ was accompanied by the formation of a key intermediate product that we identified as nitrourea by comparison with a reference material. Nitrourea is unstable in water and suffered both biotic and abiotic decomposition to eventually give NH(3), N(2)O, and CO(2). However, we were unable to detect urea. Based on products distribution and reaction stoichiometry, we suggested that degradation of NQ, O(2)NN═C(NH(2))(2), might involve initial enzymatic hydroxylation of the imine, -C═N- bond, leading first to the formation of the unstable α-hydroxynitroamine intermediate, O(2)NNHC(OH)(NH(2))(2), whose decomposition in water should lead to the formation of NH(3), N(2)O, and CO(2). NQ biodegradation was induced by nitroguanidine itself, L-arginine, and creatinine, all being iminic compounds containing a guanidine group. This first description of NQ mineralization by a bacterial isolate demonstrates the potential for efficient microbial remediation of NQ in soil.

The present study describes the use of two commercially available lignins, namely, alkali and organosolv lignin, for the removal of 2,4-dinitroanisole (DNAN), a chemical widely used by the military and the dye industry, from water. Sorption of DNAN on both lignins reached equilibrium within 10 hr and followed pseudo second-order kinetics with sorption being faster with alkali than with organosolv lignin, i.e. k2 10.3 and 0.3 g/(mg x hr), respectively. In a separate study we investigated sorption of DNAN between 10 and 40 degrees C and found that the removal of DNAN by organosolv lignin increased from 0.8 to 7.5 mg/g but reduced slightly from 8.5 to 7.6 mg/g in the case of alkali lignin. Sorption isotherms for either alkali or organosolv lignin best fitted Freundlich equation with enthalpy of formation, deltaH0 equaled to 14 or 80 kJ/mol. To help understand DNAN sorption mechanisms we characterized the two lignins by elemental analysis, BET nitrogen adsorption-desorption and 31P NMR. Variations in elemental compositions between the two lignins indicated that alkali lignin should have more sites (O- and S-containing functionalities) for H-bonding. The BET surface area and calculated total pore volume of alkali lignin were almost 10 times greater than that of organosolv lignin suggesting that alkali lignin should provide more sites for sorption. 31P NMR showed that organosolv lignin contains more phenolic -OH groups than alkali lignin, i.e., 70% and 45%, respectively. The variations in the type of OH groups between the two lignins might have affected the strength of H-bonding between DNAN and the type of lignin used.

An important R and D effort was dedicated to the characterization of ranges and training areas and to the study of the environmental fate and the ecotoxicological impacts of munitions constituents in the last 20 years in Canada and the United States. Major environmental issues were identified, and the sources of munitions constituents in training ranges are better understood. Protocols were developed for collecting representative soil samples and their effective processing. In the last years, a large effort was dedicated to the measurement of the mass of munitions constituents deposited both at target impact areas and at firing positions, which led to a good estimation of source terms of contaminants. In Canadian ranges and training areas, efforts were also dedicated to characterize both surface and subsurface aquifers and geology, and detailed hydrogeological and geological mapping. All the data acquired over the last years have been used to build hazards and vulnerability maps, which can be combined to draw risk maps that represent great assets from a risk-management perspective. The next step is the development of environmentally sound solutions that will sustain military training and maintain force readiness. In order to achieve that goal, efforts are committed to the modification of actual live-firing activities to minimize their environmental adverse impacts. Finally, Canada is aiming at developing greener and insensitive munitions that will ease the environmental pressure. This paper is a summary of what has been done in North America toward understanding and minimizing the environmental footprint of munitions.

Nitroglycerin (NG) is widely used for the production of explosives and solid propellants, and is a soil contaminant of concern at some military training ranges. NG phytotoxicity data reported in the literature cannot be applied directly to development of ecotoxicological benchmarks for plant exposures in soil because they were determined in studies using hydroponic media, cell cultures, and transgenic plants. Toxicities of NG in the present studies were evaluated for alfalfa (Medicago sativa), barnyard grass (Echinochloa crusgalli), and ryegrass (Lolium perenne) exposed to NG in Sassafras sandy loam soil. Uptake and degradation of NG were also evaluated in ryegrass. The median effective concentration values for shoot growth ranged from 40 to 231 mg kg- 1 in studies with NG freshly amended in soil, and from 23 to 185 mg kg- 1 in studies with NG weathered-and-aged in soil. Weathering-and-aging NG in soil did not significantly affect the toxicity based on 95 percent confidence intervals for either seedling emergence or plant growth endpoints. Uptake studies revealed that NG was not accumulated in ryegrass but was transformed into dinitroglycerin in the soil and roots, and was subsequently translocated into the ryegrass shoots. The highest bioconcentration factors for dinitroglycerin of 685 and 40 were determined for roots and shoots, respectively. Results of these studies will improve our understanding of toxicity and bioconcentration of NG in terrestrial plants and will contribute to ecological risk assessment of NG-contaminated sites.

2,4-Dinitroanisole (DNAN) is a low sensitive melt-cast chemical being tested by the Military Industry as a replacement for 2,4,6-trinitrotoluene (TNT) in explosive formulations. Little is known about the fate of DNAN and its transformation products in the natural environment. Here we report aerobic biotransformation of DNAN in artificially contaminated soil microcosms. DNAN was completely transformed in 8 days in soil slurries supplemented with carbon and nitrogen sources. DNAN was completely transformed in 34 days in slurries supplemented with carbons alone and persisted in unamended microcosms. A strain of Bacillus (named 13G) that transformed DNAN by co-metabolism was isolated from the soil. HPLC and LC-MS analyses of cell-free and resting cell assays of Bacillus 13G with DNAN showed the formation of 2-amino-4-nitroanisole as the major end-product via the intermediary formation of the arylnitroso (ArNO) and arylhydroxylamino (ArNHOH) derivatives, indicating regioselective reduction of the ortho-nitro group. A series of secondary reactions involving ArNO and ArNHOH gave the corresponding azoxy- and azo-dimers. Acetylated and demethylated products were identified. Overall, this paper provides the evidence of fast DNAN transformation by the indigenous microbial populations of an amended soil with no history of contamination with explosives and a first insight into the aerobic metabolism of DNAN by the soil isolate Bacillus 13G.

Triethylene glycol dinitrate (TEGDN) is an energetic plasticizer presently used in the synthesis of new insensitiveformulations. However, little is known about its environmental fate and impact. In the present study, wemeasured several environmental physicochemical parameters that are necessary to predict the fate and impact ofthe chemical in the environment. High solubility of TEGDN in water (7,430mg L1 at 25 8C), its low log Kowvalue (0.79), and low Kd values (0.09-0.78 L kg1) suggest that TEGDN will be very mobile in soils. We alsoinvestigated the kinetics and mechanisms of several degradation processes likely to occur in the environment.Hydrolysis and degradation in sterile soils were found to be very slow. No significant biological transformationwas detected after 3-month incubation in soils under aerobic conditions. Photolysis was the fastest process totake place with a rate of 0.049 day1 (t1/2¼14 days) and 0.031 day1 (t1/2¼22 days) at 258C in the dissolved ordry form, respectively. Nitrate, formaldehyde, and formate were the major photoproducts. Glyoxal, glycolaldehyde,and polycondensation products were also observed in the absence of water.

GIM (Greener Insensitive Material) is a new explosive formulation made of HMX (51.5%), TNT (40.7%), and a binder, ETPE (7.8%), which is currently investigated by the Canadian Department of National Defense for a wider use by the Army. In the present study, dissolution of GIM in water was measured and compared to the dissolution of octol (HMX/TNT: 70/30). Although the presence of ETPE did not prevent completely TNT and HMX from dissolving, GIM appeared to dissolve more slowly than octol. The ETPE was shown to prevent the formulation particles from collapsing and to retard the dissolution of both TNT and HMX by limiting their exposure to water. In both octol and GIM, the dissolution rate of the particles was governed by the compound(s) that are slower to dissolve, i.e. HMX in octol, and HMX and ETPE in GIM. A model based on Fick's diffusion law allowed fitting well the dissolution data of octol but was less appropriate to fit the data of GIM likely due to a physical rearrangement of the solid upon dissolution. The present findings demonstrate that ETPE in GIM decreases the risks of explosives leakage from particles of the new formulation and should facilitate the collecting of non-exploded GIM particles in training sites.

The wide use of nitroorganic based energetic chemicals (NOCs) such as the aromatic TNT and the non-aromatic cyclic nitramines RDX and HMX has resulted in the contamination of vast terrestrial and aquatic systems. Several reports described the toxic and carcinogenic effects of some of these chemicals and their degradation products to various terrestrial, aquatic and avian receptors. However, to validate these claims and to determine the true identity of the chemicals that causes the effect, we need to first understand the transport and transformation mechanisms of these chemicals. NOCs are a special class of chemicals with unique physical, chemical and biochemical properties making them mobile and labile molecules in the environment. Consequently, NOCs have the potential to migrate through subsurface soil and to undergo (bio)transformation to other chemicals, which raises the question "what is the real impact of these chemicals on the environment?" To be able to provide an unequivocal answer to the above question we must first understand various abiotic and biotic transformation routes that these chemicals may encounter in the environment under both aerobic and anaerobic conditions. Secondly, we need to know the ability of these chemicals and their degradation products to migrate through the environment. Knowledge of the transport and transformation routes of these chemicals will help understand their potential environmental impact.

The heterocyclic polynitramine hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a highly energetic compound found as a soil contaminant at some defense installations. Although RDX is not lethal to soil invertebrates at concentrations up to 10,000 mg/kg, it decreases earthworm cocoon formation and juvenile production at environmentally relevant concentrations found at contaminated sites. Very little is known about the uptake of RDX in earthworms and the potential risks for food-chain transfer of RDX in the environment. Toxicokinetic studies were conducted to quantify the bioaccumulation factors (BAFs) using adult earthworms (Eisenia andrei) exposed for up to 14 d to sublethal concentrations of nonlabeled RDX or [14C]RDX in a Sassafras sandy loam soil. High-performance liquid chromatography of acetonitrile extracts of tissue and soil samples indicated that nonlabeled RDX can be accumulated by the earthworm in a concentration- and time-dependent manner. The BAF, expressed as the earthworm tissue to soil concentration ratio, decreased from 6.7 to 0.1 when the nominal soil RDX concentrations were increased from 1 to 10,000 mg/kg. Tissue concentrations were comparable in earthworms exposed to nonlabeled RDX or [14C]RDX. The RDX bioaccumulation also was estimated using the kinetically derived model (BAFK), based on the ratio of the uptake to elimination rate constants. The established BAFK of 3.6 for [14C]RDX uptake was consistent with the results for nonlabeled RDX. Radioactivity also was present in the tissue residues of [14C]RDX-exposed earthworms following acetonitrile extraction, suggesting the formation of nonextractable [14C]RDX metabolites associated with tissue macromolecules. These findings demonstrated a net accumulation of RDX in the earthworm and the potential for food-chain transfer of RDX to higher-trophic-level receptors.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a common contaminant of soil and water at military facilities. The present study describes degradation of RDX with zerovalent iron nanoparticles (ZVINs) in water in the presence or absence of a stabilizer additive such as carboxymethyl cellulose (CMC) or poly(acrylic acid) (PAA). The rates of RDX degradation in solution followed this order CMC-ZVINsPAA-ZVINsZVINs with k1 values of 0.816 +/- 0.067, 0.082 +/- 0.002, and 0.019 +/- 0.002 min(-1), respectively. The disappearance of RDX was accompanied by the formation of formaldehyde, nitrogen, nitrite, ammonium, nitrous oxide, and hydrazine by the intermediary formation of methylenedinitramine (MEDINA), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine), TNX (hexahydro-1,3,5-trinitroso-1,3,5-triazine). When either of the reduced RDX products (MNX or TNX) was treated with ZVINs we observed nitrite (from MNX only), NO (from TNX only), N2O, NH4+, NH2NH2 and HCHO. In the case of TNX we observed a new key product that we tentatively identified as 1,3-dinitroso-5-hydro-1,3,5-triazacyclo-hexane. However, we were unable to detect the equivalent denitrohydrogenated product of RDX and MNX degradation. Finally, during MNX degradation we detected a new intermediate identified as N-nitroso-methylenenitramine (ONNHCH2NHNO2), the equivalentof methylenedinitramine formed upon denitration of RDX. Experimental evidence gathered thus far suggested that ZVINs degraded RDX and MNX via initial denitration and sequential reduction to the corresponding nitroso derivatives prior to completed decomposition but degraded TNX exclusively via initial cleavage of the N-NO bond(s).

Perchlorate is a uniquely stable chemical described as an emerging thyroid disrupting agent that is presently detected in several terrestrial and aquatic matrices. The present study was undertaken to deoxygenate perchlorate to the chloride anion photolytically in the presence of dodecamethylcyclohexasilane (Me2Si)6 1. It is found that photolysis of 1 in the presence of dry NaClO4 in tetrahydrofuran (THF) at 254 nm leads to the disappearance of the salt. The removal of ClO4– occurred with the concurrent formation of ClO3– and ClO2–, which disappear to eventually produce the chloride anion quantitatively. The two cyclic silanes (Me2Si)5 3 and (Me2Si)4 4 in addition to several other siloxanes that include (Me2SiO)3, (Me2SiO)4, and (Me2Si)xO2 (x = 4 and 5) were also detected. When the reaction was repeated using uniformly labelled 18O-[ClO4–] it was found that oxygen incorporated in the siloxane products was derived from perchlorate. Mixing 1 with perchlorate in THF in the dark or adding the salt to 1 after the latter being photolyzed in THF did not deoxygenate ClO4–. Based on experimental evidence gathered thus far it is concluded that dimethylsilylene, Me2Si: 2, a reactive intermediate produced by the photolysis of 1, is in part responsible for the deoxygenation of perchlorate. Direct oxygen transfer from ClO4– to the silanes during photolysis is also suggested as a potential route of deoxygenating ClO4–.

An Agilent 3DCE capillary electrophoresis system using sulfobutylether-beta-cyclodextrin (SB-beta-CD)-ammonium acetate separation buffer pH 6.9 was coupled to a Bruker Esquire 3000+ quadrupole ion trap mass detector via a commercially available electrospray ionization interface with acetonitrile sheath flow. The CE-MS system was applied in negative ionization mode for the resolution and detection of nitroaromatic and polar cyclic or caged nitramine energetic materials including TNT [2,4,6-trinitrotoluene, formula mass (FW) 227.13], TNB (1,3,5-trinitrobenzene, FW 213.12), RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine, FW 222.26) HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, FW 296.16), and CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane, FW 438.19). The CE-MS system conformed to the high-performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) and HPLC-MS reference methods for the identification of energetic contaminants and their degradation products in soil and marine sediment samples.

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) is a recalcitrant energetic chemical that tends to accumulate in soil, close to the surface. The present study describes the aerobic biodegradability of HMX using Phanerochaete chrysosporium. When added to 7 day old static P. chrysosporium liquid cultures, HMX (600 nmol) degraded within 25 days of incubation. The removal of HMX was concomitant with the formation of transient amounts of its mono-nitroso derivative (1-NO-HMX). The latter apparently degraded via two potential routes: the first involved N-denitration followed by hydrolytic ring cleavage, and the second involved alpha-hydroxylation prior to ring cleavage. The degradation of 1-NO-HMX gave the ring-cleavage product 4-nitro-2,4-diazabutanal (NDAB), nitrite (NO2 -), nitrous oxide (N2O), and formaldehyde (HCHO). Using [14C]-HMX, we obtained 14CO2 (70% in 50 days), representing three C atoms of HMX. Incubation of real soils, contaminated with either HMX (403 micromol kg(-1)) (military base soil) or HMX (3057 micromol kg(-1)), and RDX (342 micromol kg(-1)) (ammunition soil) with the fungus led to 75 and 19.8% mineralization of HMX (liberated 14CO2), respectively, also via the intermediary formation of 1-NO-HMX. Mineralization in the latter soil increased to 35% after the addition of glucose, indicating that a fungus-based remediation process for heavily contaminated soils is promising. The present findings improve our understanding about the degradation pathway of HMX and demonstrate the utility of using the robust and versatile fungus P. chrysosporium to develop effective remediation processes for the removal of HMX.

The cometabolic transformation of 2,4,6-trinitrotoluene (TNT) by an immobilized Phanerochaete chrysosporium culture was investigated under different TNT and/or glycerol feeding conditions in a 5-L reactor. In the fed-batch feeding mode, as a result of four spiking events at an average feeding rate of 20 mg TNT L(-1) d(-1) and 250 mg glycerol L(-1) d(-1), the initial TNT transformation rate and the glycerol uptake rate of the 7-day-old immobilized cell culture were 2.41 mg L(-1) h(-1) and 16.6 mg L(-1) h(-1), respectively. Thereafter, the TNT fed into the reactor depicted a negative effect on the cell physiology of P. chrysosporium, i.e., both rates decreased constantly. At 32 mg TNT L(-1) d(-1) feeding rate, also in the presence of glycerol (200 mg L(-1) d(-1)), this effect on the fungal cell metabolism was even more significant. When TNT was fed alone at 3.7 mg L(-1) d(-1), it showed an initial 0.75 mg L(-1) h(-1) rate of TNT transformation, i.e., one-third the initial level observed in the presence of glycerol. In contrast, in the continuous feeding mode (dilution rate, D = 0.11 d(-1)), at 5.5 mg TNT L(-1) d(-1) and 220 mg glycerol L(-1) d(-1), the immobilized cell culture exhibited a constant TNT transformation rate for cultivation periods of 50 and 61 days, under uncontrolled and controlled pH conditions, respectively. Thereafter, during the latter experiment, 100% TNT biotransformation was achieved at 1,100 mg L(-1) d(-1) glycerol feeding rate. Immobilized cells (115-day-old), sampled from a continuous TNT feeding experiment, mineralized [(14)C]-TNT to a level of 15.3% following a 41-day incubation period in a microcosm.

Earthworm survival tests are commonly used in terrestrial ecotoxicology to assess the toxicity of compounds in soil. Earthworm (Eisenia andrei) reproduction tests were used to assess the sublethal and chronic effects of 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5,7-triazacyclohexane (RDX). Effects on reproduction parameters (total number of cocoons, number of hatched cocoons, number of juveniles, juvenile biomass, and hatchability) were measured in TNT- and RDX-spiked artificial soil. For TNT, the lowest-observed-effect concentration (LOEC) was 110 mg/kg dry soil, and the no-observed-effect concentration (NOEC) was 55 mg/kg. For the RDX-spiked soil, the LOEC was 95 mg/kg dry soil, and the NOEC was

Native soil microbial populations and unadapted municipal anaerobic sludges were compared for nitramine explosive degradation in microcosm assays under various conditions. Microbial populations from an explosive-contaminated soil were only able to mineralize 12% hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (at a concentration of 800 mg/kg slurry) or 4% octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (at a concentration of 267 mg/kg slurry). In contrast, municipal anaerobic sludges were able to mineralize them to carbon dioxide, with efficiencies of up to 65%. Reduction of RDX and HMX into their corresponding nitroso-derivatives was notably faster than their mineralization. The biodegradation of HMX was typically delayed by the presence of RDX in the microcosm, confirming RDX is used as an electron acceptor preferentially to HMX. The laboratory-scale bioslurry reactor reproduced the results of the microcosm assays, yet with much higher RDX and HMX degradation rates. A radiolabel-based mass balance in the soil slurry indicated that, besides a significant mineralization to carbon dioxide, 25% and 31% of RDX and HMX, respectively, appeared as acetonitrile-extractable metabolites, while the remaining part was incorporated into biomass and irreversibly bound to the soil matrix. About 10% of the HMX derivatives were estimated to be chemically bound to the soil matrix, while for RDX the estimation was nil.

The biotransformation of 2,4,6-trinitrotoluene (TNT) (175 μM) by Phanerochaete chrysosporium with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization (liberated 14 CO 2 ) did not exceed 1%. A time study revealed the presence of several intermediates, marked by the initial formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT) followed by their successive transformation to several other products, including monoaminodinitrotoluenes (ADNT). A group of nine acylated intermediates were also detected. They included 2- N -acetylamido-4,6-dinitrotoluene and its p isomer, 2-formylamido-4,6-dinitrotoluene and its p isomer (as acylated ADNT), 4- N -acetylamino-2-amino-6-nitrotoluene and 4- N -formylamido-2-amino-6-nitrotoluene (as acetylated DANT), 4- N -acetylhydroxy-2,6-dinitrotoluene and 4- N -acetoxy-2,6-dinitrotoluene (as acetylated HADNT), and finally 4- N -acetylamido-2-hydroxylamino-6-nitrotoluene. Furthermore, a fraction of HADNTs were found to rearrange to their corresponding phenolamines (Bamberger rearrangement), while another group dimerized to azoxytoluenes which in turn transformed to azo compounds and eventually to the corresponding hydrazo derivatives. After 30 days, all of these metabolites, except traces of 4-ADNT and the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days. The biotransformation of TNT was accompanied by the appearance of manganese peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP activity was observed almost immediately after TNT disappearance, which was the period marked by the appearance of the initial metabolites (HADNT and ADNT), whereas the LiP activity was observed after 8 days of incubation, corresponding to the appearance of the acyl derivatives. Both MnP and LiP activities reached their maximum levels (100 and 10 U/liter, respectively) within 10 to 15 days after inoculation.

Laboratory studies were conducted to evaluate the biodegradation potential of four energetic compounds — RDX, TNT, GAP, and NC — under aerobic conditions. Soil samples collected from three sites were screened for microorganisms with the ability to degrade energetic compounds. Two bacteria were enriched and purified from RDX-contaminated soil by their ability to use RDX as a sole source of nitrogen. Using [14C]-labelled RDX, the ability of these isolates to mineralize RDX in liquid culture was verified. Bioaugmentation of RDX-contaminated soil with these isolates enhanced the rate and extent of RDX biodegradation. In contrast, although TNT is more recalcitrant and toxic, [14C]TNT mineralization activity was detected in TNT-contaminated soil. This activity was concentration dependent: in soil, as the concentration of TNT increased, mineralization decreased and was abrogated at 100 ppm TNT. Attempts to enrich and isolate from this soil organisms with the ability to mineralize [14C]TNT have as yet been unsuccessful. Although it remains unknown whether any of the soils we are working with are contaminated with GAP or NC, preliminary studies revealed that some of the soils harboured [14CJGAP and [ 14C]NC mineralization activities. Data obtained to date firmly establish the biodegradation potential of RDX under aerobic conditions, and suggest that biodegradation of TNT, GAP, and NC is also possible.